Synthesis and preliminary assessments of ethyl-terminated perfluoroalkyl nonionic surfactants derived from tris(hydroxymethyl)acrylamidomethane

Article abstract of DOI:10.1021/ol990558f

(formula presented) We describe the synthesis and preliminary physicochemical and biological assessments of a new class of nonionic hybrid hydrofluoro amphiphiles derived from tris(hydroxymethyl)aminomethane (THAM). The synthesis of the hydrophobic tail of these amphiphiles is based on the preparation of an asymmetrical hydrofluorocarbon derivative containing an ethyl segment, a fluorocarbon core, and an ethyl thiol moiety. This molecule led to either THAM galactosylated monoadducts or telomers. These amphiphiles exhibit neither detergency toward cell membranes nor membrane protein denaturation.

Full text of DOI:10.1021/ol990558f

ORGANIC
LETTERS
1
999
Vol. 1, No. 11
689-1692
Synthesis and Preliminary Assessments
of Ethyl-Terminated Perfluoroalkyl
Nonionic Surfactants Derived from
Tris(hydroxymethyl)acrylamidomethane
1
†
‡
§
§
Philippe Barth e´ l e´ my, Bruno Ameduri, Elodie Chabaud, Jean-Luc Popot, and
,†
Bernard Pucci*
Laboratoire de Chimie Bioorganique et des Syst e` mes Mol e´ culaires Vectoriels,
Facult e´ des Sciences, UniVersit e´ d’AVignon, 33 rue Louis Pasteur, 84000 AVignon,
France, Ecole Nationale de Chimie de Montpellier, UPRESA CNRS 5076,
8
rue de l’ecole Normale, F 34296 Montpellier Cedex 5, France, and Laboratoire de
Physico-Chimie Mol e´ culaire des Membranes Biologiques, CNRS UPR 9052, Institut de
Biologie Physico-Chimique and UniVersit e´ Paris-7, 13 rue Pierre et Marie Curie,
F-75005 PARIS, France
bernard.pucci@uniV-aVignon.fr
Received April 6, 1999
ABSTRACT
We describe the synthesis and preliminary physicochemical and biological assessments of a new class of nonionic hybrid hydrofluoro amphiphiles
derived from tris(hydroxymethyl)aminomethane (THAM). The synthesis of the hydrophobic tail of these amphiphiles is based on the preparation
of an asymmetrical hydrofluorocarbon derivative containing an ethyl segment, a fluorocarbon core, and an ethyl thiol moiety. This molecule
led to either THAM galactosylated monoadducts or telomers. These amphiphiles exhibit neither detergency toward cell membranes nor membrane
protein denaturation.
Stabilizing membrane proteins in aqueous solution under
their native state is a difficult task. The lipids and proteins
that make up biological membranes classically can be
dispersed using detergents. The hydrophobic moiety of
detergents competes with lipids for the transmembrane,
hydrophobic surface of the proteins and thereby renders them
water-soluble. Substituting detergent for lipids however
frequently inactivates the protein. While the mechanism of
this inactivation generally is not known, it is likely to involve
either the removal of critical lipids and/or a direct perturba-
tion by the detergent of the protein’s structure. To alleviate
this problem, we are exploring the use of less dissociating
1
,2
molecules, namely fluorinated surfactants. While highly
tensioactive, perfluorinated surfactants are not detergents and
1
do not solubilize biological membranes. Some of them,
nevertheless, have proven able to keep water-soluble the
membrane proteins that had been extracted using a classical
2
hydrogenated detergent. In the course of this previous study,
we noted however that perfluorinated molecules that exhib-
(1) Maurizis, J. C.; Pavia, A. A.; Pucci, B. Biorg. Med. Chem. Lett. 1993,
3
, 161-164. Pucci, B.; Maurizis, J. C.; Pavia, A. A., Eur. Polym. J. 1991,
†
Universit e´ d’Avignon.
Ecole Nationale de Chimie de Montpellier.
Institut de Biologie Physico-Chimique and Universit e´ Paris-7.
27, 1101-1106.
‡
(2) Chabaud, E.; Barth e´ l e´ my, P.; Mora, N.; Popot, J. L.; Pucci, B.
Biochimie 1998, 80, 515-530.
§
1
0.1021/ol990558f CCC: $18.00 © 1999 American Chemical Society
Published on Web 10/24/1999

ited this interesting property were rare and that, somewhat
predictably, they had difficulty preventing the proteins from
2
aggregating. In the present work, we describe the synthesis
and preliminary characterization of hydrofluorinated surfac-
tants. These molecules feature a largely fluorinated alkyl
chain terminated by an ethyl group. The rationale behind
their design is that the ethyl group would improve the affinity
of the tip of the tail for the hydrogenated surface of the
protein, while doing little to restore detergency.
More than three decades ago, Brace reported that long-
chain alkanoic acids bearing perfluoroalkyl terminal segments
3
had unique surface active and wettability properties. Since
then, numerous works have described the potentialities of
hydrofluorocarbon compounds. Hybrid anionic surfactants
containing fluorocarbon and hydrocarbon chains have already
4
been prepared and studied. Such hybrid compounds tend
5
to form supramolecular assemblies, some of which have
6
potential biological applications. Recently, hybrid bola-
Figure 1. Synthesis of the hydrofluorocarbon thiol 3. Conditions:
a) C , CuI, 165 °C, 15 h, yield 80%; (b) AIBN, tri-n-butyltin
hydride, THF, ∆, 6 H; (c) the residual diiodo is reused; the yield
rises to 60%; (d) thiourea, THF/H
at room temperature, yield 95%.
amphiphiles have been synthesized to study the “flip-flop”
(
2 4
H
7
behavior of spin label in vesicles. Few articles however have
described the synthesis of perfluoro compounds with an alkyl
2 4
O 20/1 ∆, followed by NH OH
8
terminal part. Among them, one can point out the work
achieved by Rondestvedt who prepared methyl-terminated
perfluoroalkyl iodides (from telomerization reactions). Such
compounds can provide very useful starting materials. So
initiated with azobis(isobutyronitrile) (AIBN). This step
affords monoiodo, diiodo, and a very small amount of bis-
hydrogenated compounds. Diiodo compound 1 can be easily
recovered by rapid chromatography on silica gel (eluent:
hexane) and reused to complete the reaction. 1-Iodo-
9
far, only a handful of hydrofluoro surfactants have been
1
0
prepared.
The work reported here deals with the synthesis and
preliminary physicochemical assessment of new ethylfluoro-
carbon surface-active molecules derived from galactosylated
tris(hydroxymethyl)acrylamidomethane.
3
,3,4,4,5,5,6,6,7,7,8,8-dodecafluorodecane 2 reacts with the
thiourea to give, after hydrolysis, thiol derivative 3 (Figure
).
Since hydrofluorocarbon thiol 3 is now available, the
1
Results and Discussion. 1. Synthesis. The key synthetic
task was to obtain a nonsymmetrical fluorocarbon derivative
containing a distal alkyl segment, a fluorocarbon core, and
a proximal ethyl thiol moiety. This molecule will be the
hydrophobic tail of the surfactant. Such a building block is
not commercially available. To open a new route toward
mixed nonionic amphiphiles, symmetrical R,ω-diiodoper-
fluorohexyl was chosen as a starting material.
preparation of novel nonionic ethyl-terminated fluorocarbon
surfactants becomes feasible. Basically, the thiol derivative
opens an access to a new class of nonionic surfactants, which
can be either monoadducts or amphiphile telomers.
Several telomers derived from the THAM (tris(hydroxy-
methyl)acrylamidomethane) bearing hydrocarbon or fluoro-
carbon tails have already been prepared; however, none of
As previously described,11 the bis-monoethylenation of
diiodoperfluorohexane leads to the expected starting material
12
them carry a mixed hydrofluoro chain.
The telomerization reaction was performed in the presence
of AIBN and thiol 3 as chain-transfer reagent (Figure 2).
Telomers were isolated by precipitation in methanol-ether
mixture (10/90, v/v). The byproducts, mainly the monoad-
duct, were soluble in this solvent.
1
(
,10-diiodo-3,3,4,4,5,5,6,6,7,7,8,8-dodecafluorodecane
Figure 1).
The second step involves the selective reduction of one
1
iodine only achieved by tri-n-butyltin hydride. The outcoming
radical is trapped by tin hydride providing a chain reaction
Unlike the usual fluorocarbon telomers, the average degree
n
of polymerization (DP ) of these compounds can be deter-
(
(
(
3) Brace, N. O. J. Org. Chem. 1962, 27 (12), 4491-4498.
4) Yoshino, N.; Hamano, K.; Omiya, Y. Langmuir 1995, 11, 466-469.
5) Guedj, C.; Pucci, B.; Zarif, L.; Coulomb, C.; Riess, J. G.; Pavia, A.
A. Chem. Phys. Lipids 1994, 2, 153-173.
6) Clary, L.; Santaella, C.; Vierling, P. Tetrahedron 1995, 51, 13073-
3088.
7) Yu, B.; Hui, Y. Supramol. Chem. 1995, 5, 193-195. Liang, K.; Hui,
Y. J. Am. Chem. Soc. 1992, 114, 6588-6590.
8) Szlavik, Z.; Csampai, A.; Krafft, M. P.; Riess, J. G.; Rabai, J.
(
1
(
(
Tetrahedron Lett. 1997, 38, 8757-8760.
9) Rondestvedt, C. S. J. Org. Chem. 1977, 42, 1985-1990.
1
997, 38, 1937-1940. Hu, C. M.; Qing, F. L. J. Org. Chem. 1991, 56,
6
348-6351.
(11) Manseri, A.; Ameduri, B.; Boutevin, B.; Kotora, M.; Hajeck, M.;
Figure 2. Hydrofluorocarbon telomer (DP
n
) 10).
Caporiccio, G. J. Fluorine Chem. 1995, 73, 151-158.
1690
Org. Lett., Vol. 1, No. 11, 1999

1
mined more easily by H NMR spectroscopy. Indeed, since
The aim of this study is to examine the physicochemical
data obtained with our ethyl perfluorocarbon nonionic
surfactants. The results are compared to those measured with
hydrogenated and/or fluorinated homologues previously
described.
the integration of the terminal methyl protons resonance is
measured accurately, the DP value becomes reliable. It was
n
worthwhile to prepare such a hydrofluorocarbon telomer by
using similar ratio thiol/THAM as perfluorocarbon com-
n
pounds to eventually verify and confirm the DP previously
measured. (One can assume that the transfer constant is quite
equivalent for both telogen agents.)
The CMC data of ethyl-terminated perfluoro nonionic
surfactants were determined following the usual procedure
by measuring their surface tension properties. The CMC’s
and limit tension results obtained are listed in Table 1.
According to the thiol/THAM ratio chosen, the THAM
monoadduct can be obtained and isolated after silica gel
chromatography (eluent: MeOH/AcOEt, 10/90, v/v). How-
ever, the compound bearing a trishydroxyl head shows a poor
solubility in water. Obviously, such a property is required
to get efficient amphiphiles. Thus, sugar moieties were
grafted on the hydroxyl groups to increase the water
solubility of the whole molecule. The THAM galactosylated
monoadducts were then prepared under free-radical condi-
Table 1. Physicochemical Data of Surfactants Bearing
Ethylfluorocarbon, Hydrofluorocarbon, and Perfluorocarbon
Tails
product
no.
CMC,
mMd
γ limit,
mN/m
P
13
tions following the procedure previously described (Figure
). For example, main physicochemical data of galactosylated
tetraacetyl) surfactant 4a are reported in full note 20.
C2H5(C6F12)CH2CH2-
STHAM TriGal
C2H5(C6F12)CH2CH2-
STHAM DiGal
C2H5(C6F12)CH2CH2-
STHAM MonoGal
C2H5(C6F12)CH2CH2-
S(THAM)2 TriGal
C2H5(C6F12)CH2CH2-
STeloTHAM, DPn ≈ 10
C10H21- Telo, DPn ) 5,7
C12H25- Telo, DPn ) 4
C8F17CH2CH2- Telo, DPn ) 6
C8F17CH2CH2STHAM TriGal
C8F17CH2CH2STHAM DiGal
C6F13CH2CH2STHAM TriGal
C10H21STHAM TriGal
C12H25STHAM DiGal
C12H25STHAM TriGal
4a
4b
4c
4d
4e
0.5
25/26
26
3
(
0.35
0.3
25/26
25/26
32
0.35
0.45
5c
0.6
6b
0.15
0.03
0.038
0.046
0.28
1
31.3/32
25/27
27
25
34
7b
8b
9b
10b
11b
12b
13b
0.28
0.23
35
45
a
Telo: telomer. b See ref 13. c See ref 1. d CMC were measured with a
tensiometer Kruss 2000.
Figure 3. Synthesis of sugar derivatives. Substituents: R
Gal(OAc) or H; R , R , â-D-Gal or H. Conditions: (a) AIBN,
MeOH, 65 °C; (b) MeONa (catalytic), MeOH, 20 °C. 4a ) R
: â-D-galactose; 4b ) R : H; R : â-D-galactose; 4c ) R , R
H; 4d ) R , R : â-D-galactose, diadduct.
1
, â-D-
4
2
3
As observed previously for perfluoro- or hydrocarbon
THAM derivatives (telomers, monoadducts; see Table 1),
the CMC of hydrofluorocarbon surfactants depends es-
sentially on the hydrophobic part of the molecule. Indeed,
no drastic changes of CMC values are observed when the
hydrophilic head volume increases (CMCtelo 4e, 0.45 mM;
CMCtrigal 4a, 0.5 mM; CMCmonogal 4c, 0.3 mM). Since the
CMC depends essentially on the nature and the length of
the tail, it was reasonable to expect, in the case of ethylfluo-
rocarbon compounds, a CMC close to that of perfluoro
2
,
R
3
2
3
2
3
:
2
3
2. Physicochemical Properties. Surfactants containing a
fluorocarbon chain as hydrophobic group display high surface
activity and low CMC (critical micelle concentration).
Recently, it was shown that the hydrophobicity is higher in
the case of perfluorinated ionic surfactants than their
hydrogenated analogues, suggesting that the mechanism of
the micellization process is similar.14 Hybrid surfactants
bearing hydro- and fluorocarbon tails are quite unknown in
terms of tensioactive properties.
analogues 8 or 9, which bear a C
0.04mM). Actually, the CMC measured is 10 times higher
even though it remains lower than that of the corresponding
8
F
17CH
2
CH
2
tail (CMC ≈
hydrogenated compounds (11: tail, C10H21; CMC, 1mM).
Free energies of micellization can be evaluated from the
1
5
(
12) Pucci, B.; Pavia, A. A. Tetrahedron Lett. 1991, 32, 1437-1440.
CMC. They show that micelles form more favorably in
the case of ethylfluorocarbon compounds (C -,
Pavia, A. A.; Pucci, B.; Riess, J. G.; Zarif, L. Bioorg. Med. Chem. Lett.
2 5 6 12 2 4
H C F C H
1
991, 1, 103-106.
13) Polidori, A.; Pucci, B.; Maurizis, J. C.; Pavia, A. A., New. J. Chem.
994, 18, 839-848.
14) Tomasic, V.; Chittofrati, A.; Kallay, N. Colloids and Surfaces, A:
(
∆G ≈ -19 kJ) than for hydrocarbon surfactants (C10
H21-,
1
(
Physicochem. Eng. Aspects 1995, 104, 95-99.
(15) Berthod, A. J. Chim. Phys. 1983, 80, 407-423.
Org. Lett., Vol. 1, No. 11, 1999
1691

∆
G ≈ -16.7 kJ) and less easily than for their hydrogenated
examine the ability of hybrid surfactants to keep membrane
proteins in solution under their native state. Cytochrome b
f is highly sensitive to denaturation by classical detergents
when those are used in the absence of protective lipids.19
16
analogues (C
F
8 17
C
2 4
H -, ∆G ≈ -25 kJ).
6
It was found previously that for a given length of the
fluorinated chain (in a nonionic surfactant series), increasing
the length of the hydrogenated spacer (hydrocarbon moiety
located between the polar head and the fluorinated tail) did
not lower the CMC values as much as could have been
expected. This may indicate that the length of the fluori-
nated chain controls the micellization phenomenon, the
hydrocarbon part (as a spacer) playing only a minor role. In
the present work, the grafting of a hydrocarbon moiety at
the end of the fluorocarbon tail seems to destabilize the
micelle organization, i.e., the packaging of fluorocarbon chain
in the micelle core, and thus increases their CMC. This would
suggest that interactions between hydrocarbon and fluorinated
parts in the core of the micelles are unfavorable.
6
Following transfer of purified cytochrome b f to lipid-free
solutions of either compound 4a or 4e according to the
sucrose gradient procedure described in ref 2, the complex
remained water-soluble, monodisperse, intact (with regard
to its subunit and pigment composition), and enzymatically
active. These observations bode well for the usefulness of
hydrofluorocarbon surfactants in membrane protein bio-
chemistry.
1
7
Acknowledgment. This research was supported in part
by the interdisciplinary program Physique et chimie du
Vivant and the groupement de recherche Colloides en
Interaction of the Centre National de la Recherche Scientifique.
3. Biochemical Properties. Biochemical tests conducted
as described in ref 2 showed that neither of our hybrid
surfactants exhibited any detergency. Specifically, none of
them extracted integral membrane proteins from thylakoid
membranes from the unicellular alga Chlamydomonas re-
inhardtii. These observations suggest that, as expected,
hybrid hydrophobic tails show too low a partition coefficient
into lipid bilayers for a disruptive concentration to be
OL990558F
(
18) Cramer, W. A.; Soriano, G. M.; Ponomarev, M.; Huang, D.; Zhang,
H.; Martinez, S. E.; Smith, J. L. Annu. ReV. Plant Physiol. Plant Mol. Biol.
996, 47, 477-508.
19) Breyton, C.; Tribet, C.; Olive, J.; Dubacq, J.-P.; Popot, J.-L. J. Biol.
Chem. 1997, 272, 21892-21900.
1
(
1
(
20) HFTHAMtri(GalOAc4) 4a: NMR H (CDCl3) δ (in ppm) 1.07 (t,
3
3
4
H, JHH ) 7.5 Hz, CH3), 2.1 (m, 4s, 14H, CH3CH2, 3(CH3CO)4), 2.4 (m,
H, CH2CO, CF2CH2), 2.8 (m, 4H, CH2SCH2), 3.7 (m, 2H, H6), 4.16 (m,
reached. Cytochrome b
plastocyanin oxidoreductase from oxygenic photosynthesis,
was used as a convenient model system with which to
6
f complex, the plastoquinol-
18
9H, H5, 3(CH2O), 4.5 (d, 3H, H1), 5.05 (dd, 3H, H2), 5.15 (dd, 3H, H3),
5
.5 (m, 3H, H4), 6.1 (s, 1H, NH); NMR 13C (CDCl3) δ (in ppm) 4.8 (t,
3
2
JCF, ) 5 Hz, CH3), 20.5, 20.7 (CH3CO), 23, 5 (CH2S), 25.6 (t, JCH )
2
2
4.5 Hz, CH3CH2CF2), 25, 8 (CH2S), 33.1 (t, JCH ) 24.4 Hz, CH2CF2),
36.9 (CH2CO), 61.2 (C(CH2)3), 63.0 (C6), 67.1 (C4), 68.5 (CH2Ogal), 69.2
(C2), 70.5 (C5 or C3), 70.9 (C5 or C3), 101.5 (C1 â anomer), 170.1 (COCH3),
172.7 (CO); NMR 19F (CDCl3) δ (in ppm) -114, 8 (2F, CF2CH2), -116,
8 (2F, CF2CH2), -122, 2 (4F, CH2CF2CF2), -124.0 (4F, CF2CF2CF2).
(
16) Kissa, E. Fluorinated Surfactants. Synthesis, Properties, Applica-
tions; Surfactants Science Series 50; Dekker: New York 1994
17) Sadtler, V. M.; Jeanneaux, F.; Krafft, M. P.; Rabai, J.; Riess, J. G.
New J. Chem. 1998, 22, 609-613.
(
1692
Org. Lett., Vol. 1, No. 11, 1999